Adenosine Monophosphate Binding Stabilizes the KTN Domain of

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AMP binding stabilizes the KTN domain of the Shewanella denitrificans Kef potassium efflux system Christos Pliotas, Samuel C Grayer, Silvia Ekkerman, Anthony KN Chan, Jess Healy, Phedra Marius, Wendy Bartlett, Amjad Khan, wilian augusto cortopassi, Shane A Chandler, Tim Rasmussen, Justin LP Benesch, Robert S. Paton, Timothy D. W. Claridge, Samantha Miller, Ian Rylance Booth, James H. Naismith, and Stuart J. Conway Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00300 • Publication Date (Web): 28 Jun 2017 Downloaded from http://pubs.acs.org on June 30, 2017

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Biochemistry Nucleotide Binding to Kef

AMP binding stabilizes the KTN domain of the Shewanella denitrificans Kef potassium efflux system

Christos Pliotas,a,† Samuel C. Grayer,b,† Silvia Ekkerman,c,† Anthony K. N. Chan,b,† Jess Healy,b,‡ Phedra Marius,a Wendy Bartlett,c Amjad Khan,b Wilian A. Cortopassi,b Shane A. Chandler,d Tim Rasmussen,c Justin L. P. Benesch,d Robert S. Paton,b Timothy D. W. Claridge,b Samantha Miller,b Ian R. Booth,c,* James H. Naismitha,e,f,g,* and Stuart J. Conwayb,h,*

a

Biomedical Sciences Research Complex, University of St Andrews, North Haugh, St

Andrews, KY16 9ST, UK b

Department of Chemistry, Chemistry Research Laboratory, University of Oxford,

Mansfield Road, Oxford, OX1 3TA, UK c

School of Medicine, Medical Sciences and Nutrition, Foresterhill, Aberdeen, AB25

2ZD, UK d

Department of Chemistry, Physical & Theoretical Chemistry Laboratory, University

of Oxford, South Parks Road, Oxford, OX1 3QZ, UK e f

Biotherapy Centre, Sichuan University, Chengdu, China.

RCaH, Rutherford Appleton Laboratory, Harwell Oxford, Didcot, OX11 0FA.

g

Division of Structural Biology, University of Oxford, Henry Wellcome Building for

Genomic Medicine, Old Road Campus, Roosevelt Drive, Headington, Oxford OX3 7BN. h

Freiburg Institute for Advanced Studies – FRIAS, Albert-Ludwigs-Universität

Freiburg, Albertstr. 19, 79104 Freiburg, Germany.

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ACS Paragon Plus Environment

Biochemistry

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Page 2 of 54 Nucleotide Binding to Kef

†

These authors contributed equally to this work.

‡

Current address: Department of Pharmaceutical and Biological Chemistry, UCL

School of Pharmacy, University College London, 29/39 Brunswick Square, WC1N, 1AX, UK *To whom correspondence should be addressed. Stuart Conway: [email protected]; +44(0)1865 285109. James Naismith: [email protected]; +44(0)1235567701. Ian Booth: [email protected]; +44 (0)1224 437396.

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Abstract Ligand binding is one of the most fundamental properties of proteins. Ligand functions fall into three basic types: substrates, regulatory molecules, and co-factors essential to protein stability, reactivity, or enzyme-substrate complex formation. The regulation of potassium ion movement in bacteria is predominantly under the control of regulatory ligands that gate the relevant channels and transporters, which possess subunits or domains that contain Rossmann folds (RFs). Here we demonstrate that AMP is bound to both RFs of the dimeric bacterial Kef potassium efflux system (Kef), where it plays a structural role. We conclude that AMP binds with high affinity ensuring that the site is fully occupied at all times in the cell. Loss of the ability to bind AMP, we demonstrate, causes protein, and likely dimer, instability and consequent loss of function. Regulation of Kef system function is via the reversible binding of comparatively low affinity glutathione-based ligands at the interface between the dimer subunits. We propose this interfacial binding site is itself stabilised, at least in part, by AMP binding.

Keywords Biophysical studies, crystallography, protein dimers, membrane protein, nucleotide

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A fundamental property of proteins, by which they express their function in the cell, is the binding of ligands, usually ions or molecules of small mass relative to that of the protein itself. At least three different roles are ascribed to the binding of ligands: firstly, the ligand is a substrate or essential cofactor for an enzyme; secondly, ligand binding may be purely regulatory bringing about changes in protein activity; and thirdly ligands may stabilize a protein fold. The roles are not mutually exclusive and can be combined. The activation of ligand-gated channels is usually brought about by an allosteric transition upon ligand binding at a point distant from the pore; the changes in the concentration of the ligand may reflect the biological state of either the cell or the environment. Regulation of ion flow is critical throughout biology and, for potassium ions (K+), ligand-gated channels and transporters are central to the modulation of cellular K+ pools.

In bacteria, both K+ channels, which play an important role in K+ influx, and K+ efflux systems that control the response of the bacterial cell to electrophiles, have K+ transport and NAD-binding (KTN) domains. These domains may be covalently attached to the pore or may be separately expressed entities that form non-covalent associations with the pore. Some are hybrid systems utilizing a combination of KTN domains that are part of the integral pore-forming subunit together with KTN domains expressed separately from an internal start codon on the same mRNA as the pore protein.1,2 Another major structural variation is that for the channels (e.g. TrkAH, KtrAB and MthK) octameric rings of KTN domains modulate ion flux, whereas for the Kef systems, dimeric assemblies dominate the known architectures. A conserved feature of KTN domains is a Rossmann fold, a feature known to be

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associated with nucleotide binding since its first identification in NAD(H)-binding lactate dehydrogenase.3

In Escherichia coli, and most other bacteria, multiple transport systems and channels effect control over the K+ pool, including the Kef systems, which are gated by glutathione (GSH) and its electrophilic conjugates (GSX). In Gram-positive bacteria similar protective systems might exist which employ electrophilic conjugates of other species-specific thiols,4 for example bacillithiol.5-7 Whereas the activity of most K+ transport systems causes modulation of the cytoplasmic pH in the alkaline direction, the Kef systems cause acidification in response to cell-damaging electrophiles.8,9 The ~600 residue Kef proteins form dimers of an ~380 amino acid membrane domain, which may contain up to 12 trans-membrane spans, although these are poorly defined from a structural perspective. A short hydrophilic linker (20-26 amino acids) connects the membrane domain to two further domains: an ~150 residue KTN domain, and a further, less well-conserved, domain of variable length at the extreme C-terminus of the protein. The KTN domains of separate proteins dimerize and the interface between them contains the GSH binding site. Gating of the K+ efflux system requires GSH/GSX ligand-mediated communication between the C-terminal domains and the loop containing the ion flow regulating HALESDIEP sequence.10

The Kef proteins can be broadly divided into two classes, those including E. coli KefC and KefB that require an ancillary protein (KefF11 and KefG for KefC and KefB, respectively) for full function and those, such as Shewanella denitrificans, that do not require an ancillary protein. Gating by GSH/GSX is thought to be almost identical in both protein types. Residues in the predicted GSX binding site of S.

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denitrificans Kef, identified by sequence alignment and modeling, will likely play similar roles to their E. coli KefC counterparts, which were identified by molecular genetic studies and crystallography. The E. coli KefC protein has been difficult to study biochemically due to the instability of the KefF-KefC complex, thus our studies have focused on the simpler S. denitrificans Kef protein.12

The Rossmann folds of the KTN domains in the K+ uptake systems, TrkAH and KtrAB, have been studied biochemically and shown by crystallography to bind ATP and NADH.13 These uptake systems have K+-permeable pores with pseudo-four-fold symmetry to create a pore reminiscent of the classical P-type K+ channels. Twin pores, arising from separate membrane proteins in the dimer, form associations with an octameric assembly of KTN domains. Binding of ATP and/or NADH modulate the conformation of the octameric rings and regulate the opening of the K+-conducting pore. ADP and NAD+ activate the GsuK potassium channel, via its KTN-related RCK domains, whereas Ca2+ serves as an allosteric inhibitor.14 In contrast, the structural basis of regulation of other KTN domain-regulated K+ channels (e.g. CglK, Kch, MthK) by nucleotides is poorly understood; although reversible gating by divalent cations has been described for MthK, the role of nucleotide binding in the RF is unknown.15 Similarly, for the GSX-gated Kef systems the role of the bound nucleotide is unclear.

Previously obtained X-ray crystal structures of the KTN domain of trkA from Methanocaldococcus jannaschii and the KTN domain of ktrA from Bacillus subtilis have

electron

density

consistent

with

NADH

bound

in

the

Rossmann

fold.{Roosild:2002tc} Based on these data a homology model was constructed that

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had NADH modelled into the Rossmann fold of Kef the E. coli KefFC KTN domain.{Roosild:2002tc} In subsequent crystallographic studies on the nucleotide pocket of KTN domain of the E. coli KefFC KTN domains,10 it is suggested that NADH occupies the nucleotide-binding pocket based on the homology model and the presence of this nucleotide in the crystallization liquor, although density only exists that is consistent with a bound AMP molecule. Subsequently, when structures with GSH and GSX were solved AMP was modeled into the GSH structure, but no density consistent with a nucleotide was observed in the GSX structure (density consistent with sulfate ions was observed in the RF of the GSX structure).16 The uncertainty over the identity of the bound ligand, and the lack of any insight into the role of the bound nucleotide prompted us to re-examine the system in more detail.

Here we report the structural analysis of the KTN domain from S. denitrificans. We have established that both the isolated ligand-binding C-terminal domain (SdKefCTD) and full-length integral membrane protein (SdKef) contain AMP when purified after over-expression in E. coli. Differential scanning fluorimetry (DSF, also known as thermal shift) analysis shows that incubation of SdKefCTD with additional AMP results in major stabilizing effects on the protein. NADH although unable to displace AMP from the KTN domain results in some stabilization in DSF studies. In the isolated soluble SdKefCTD KTN domain, introduction of mutations predicted to affect AMP binding led to lower protein expression, consistent with a role for AMP in protein structural integrity. In agreement with this proposal, molecular dynamics simulations indicate reduced stability of the SdKefCTD domain when AMP was removed. In the full-length protein, the same mutations yielded inactive channels. We

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propose that AMP is integral to the KTN domain in SdKef and is required for the stable and functional Kef dimer complex.

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Materials and methods Materials n-Dodecyl-β-D-maltopyranoside

(DDM)

was

purchased

by

Anatrace

(www.affymetrix.com). Glutathione (reduced) GSH, adenosine monophosphate (AMP), nicotinamide adenine dinucleotide (NAD+) and reduced nicotinamide adenine dinucleotide (NADH) were ordered from Fisher. Reagents for buffer and other chemicals were purchased from Sigma unless otherwise stated.

Expression and purification of Kef The KTN construct, denoted as SdKefQCTD, has been characterized previously and contains residues 391-607 of the full length SdKef protein, including the KTN domain, the carboxy-terminal peripheral domain, the highly charged Q-linker connecting the SdKefQCTD with the transmembrane domains and a peptide corresponding to the regulatory HELEVDIEP loop, plus a C-terminal LEH6 tag.12,25 The SdKefQCTD construct was transformed into the E. coli strain BL21(DE3) (www.bioline.com). Cells were grown in 500 mL of LB medium at 37 °C to an OD600nm ≈ 0.8. The cultures were cooled to 25 °C and induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 4 h. The cell pellet was resuspended in lysis buffer, 50 mM Tris-HCl buffer, pH 7.8, 300 mM KCl, 40 mM imidazole, 10% glycerol and 1 mM benzamidine. After disruption of the cells with a French press at 18 000 psi, the suspension was centrifuged at 4 000 g for 20 min to remove cell debris. The supernatant was then centrifuged at 100 000 g for 1 h. The supernatant was then filtered using 0.45 µm diameter filters and was passed through a 25 mL column containing 0.5 mL of nickel-nitrilotriacetic acid (Ni2+-NTA) agarose, at 4 °C. The column was washed with 15 mL of wash buffer, 50 mM Tris-HCl buffer, pH 7.8,

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300 mM KCl, 50 mM imidazole, 10% glycerol and 1 mM benzamidine, to remove non-specifically bound proteins and was left at 4 °C overnight. Next morning the elution followed with 10 mL of elution buffer, 50 mM Tris-HCl buffer, pH 7.8, 300 mM KCl, 300 mM imidazole and 0.5 mL fractions were collected. The fractions were analyzed by SDS−PAGE and UV/vis absorption spectroscopy and the fractions with the highest protein content from the Ni2+-NTA column were applied to a 120 mL Superose 6 column (General Electrics Healthcare) equilibrated with buffer containing 50 mM Tris-HCl, pH 7.8 and 300 mM KCl. Protein was then eluted at 1 mL/min flow rate. Protein concentration was monitored by absorption at 280 nm. The column was calibrated with Biorad standards. Identity and integrity were confirmed by mass spectrometry (Figure S3).

Alternative protocol The E. coli strain, MJF373,12 was used to express the SdKefQCTD protein construct, which is encoded in the pTrcSdKefQCTDH6 plasmid.12 The expression of the SdKefQCTD protein is inducible by addition of IPTG. For recombinant protein expression, E. coli MJF373 host was first transformed with the pTrcSdKefQCTDH6 plasmid. The resultant transformant was aerobically cultured in the 2× TY medium [16 g/L OxoidTM Tryptone (Thermo Fisher Scientific Inc.), 10 g/L OxoidTM Yeast Extract (Thermo Fisher Scientific Inc.), 5 g/L NaCl] at 30 °C with an agitation speed of 180 r.p.m., in the presence of 100 µg/mL of ampicillin (Apollo Scientific Ltd.). When solid medium is required, Bacto™ Agar (BD) was added to a final concentration of 1.5% (w/v) in the 2× TY medium. When the bacterial culture reached an optical density of 1.0 at 600 nm IPTG (Apollo Scientific Ltd.), was added to a final concentration of 0.8 mM to induce expression of the recombinant protein. Bacterial

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cell pellets were then harvested by centrifugation (Rotor: F10BCI-6x500y, Avanti™ J-25 Centrifuge, Beckman Coulter Inc) at 11305 g and 4 °C after 4h of IPTG postinduction, and kept at –80 °C until protein purification. The SdKefQCTD protein was subsequently purified by immobilized metal affinity chromatography (IMAC) and then size-exclusion chromatography (SEC) at 4 °C.

To prepare a sample for protein purification, the frozen cell pellet (7.70 g) was first resuspended in Extraction Buffer. Extraction Buffer was prepared by completely dissolving one tablet of SigmaFASTTM Protease Inhibitor Cocktail Tablet, EDTA Free in 100 mL of solution containing 50 mM NaH2PO4/Na2HPO4, 500 mM NaCl, 10% glycerol, pH 7.4. A hundred milliliter of Extraction Buffer per 20 g of cell mass was used for resuspension. After complete resuspension of the bacterial pellets, the cells were lysed on ice by sonication (50% amplitude, 5 s bursts interrupted by 5 s pauses for 60 cycles; Ultrasonic Processor, Sonics & Materials, Inc.) to release cytosolic proteins. Polyethyleneimine at a final concentration of 0.15% (v/v) was added from 5% (v/v) stock solution (pH 7.4) to the cell lysates and the mixture was incubated on ice for 15 min to precipitate DNA. Insoluble cell debris and precipitated DNA were removed by centrifugation (Rotor: JA25.50, Avanti™ J-25 Centrifuge, Beckman Coulter Inc.) at 25 000 g and 4 °C for 15 min. The resulting supernatant was collected and filtered through 0.45 µm pore size syringe filters (Merck Millipore Corp.). The clarified cell lysate was diluted to 100 mL with Extraction Buffer, and then added with 20 mM imidazole (Sigma-Aldrich Co.) and 10 mM β-mercaptoethanol (Bio-Rad Laboratories, Inc) at final concentrations. This cell lysate preparation was used for the first step of protein purification by IMAC.

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For protein purification by IMAC, a HisTrap HP 5 mL column (GE Healthcare) was used to purify hexa-histidine-tagged SdKefQCTD. The affinity purification columns were connected to a computerized ÄKTAFPLC system (GE Healthcare). To equilibrate the column for IMAC, 10 column volumes (CV) of Binding Buffer (50 mM NaH2PO4/Na2HPO4, 500 mM NaCl, 10% Glycerol, 10 mM βmercaptoethanol, pH 7.4) were used. The pretreated cell lysates (100 mL; from the above sample preparation procedure) were then loaded into the equilibrated HisTrap HP column. Following the sample loading into the column, Binding Buffer and Elution Buffer (50 mM NaH2PO4/Na2HPO4, 500 mM NaCl, 10% glycerol, 500 mM imidazole, pH 7.4) were mixed in different ratios to wash out non-specifically bound proteins and elute the protein of interest. Firstly, 10 CV of a step gradient containing 9% Elution Buffer (with 45 mM imidazole) and then a linear gradient ranged from 9% to 30% Elution Buffer (containing up to 150 mM imidazole) over 10 CV were used to wash out contaminating binders. For elution of the polyhistidine-tagged SdKefQCTD protein, 5 CV of 60% Elution Buffer (containing 300 mM imidazole) were used to elute the target in 2 mL fractions.

After the IMAC purification step, SdKefQCTD was further purified by SEC. The IMAC-purified SdKefQCTD protein was first concentrated by using VivaspinTM sample concentrator (GE Healthcare). The concentrated protein sample (2 mL) was then loaded via a 2 mL injection loop into a gel filtration column XK 16/70 (GE Healthcare) packed with 120 mL of Superdex 75 resin (GE Healthcare). This SEC column was pre-equilibrated with 150 mL degassed SEC Buffer (50 mM NaH2PO4/Na2HPO4, 150 mM NaCl, pH 7.4). The SEC procedure was operated at a constant flow rate (1 mL/min) over 150 mL of a total flow volume and filtrates were

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collected in 5-mL fractions (the sample collection was started at 20th mL and stopped at 120th mL). The SEC-purified protein was further concentrated by VivaspinTM sample concentrator (GE Healthcare) after the purification process. Protein concentrations were determined by a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific Inc.).

The chemicals, NaH2PO4 and Na2HPO4, were purchased from Alfa Aesar and BDH Chemicals Ltd., respectively. Glycerol and NaCl were bought from Thermo Fisher Scientific Inc. All other chemicals were purchased from Sigma-Aldrich Co. unless otherwise specified. All of the buffers used in the purification procedures were prefiltered through 0.2-µm pore size filter papers (Sartorius UK Ltd.) under vacuum to remove insoluble precipitates.

Western blot of membrane and soluble fractions containing full-length SdKef or SdKefQCTD. Membrane and soluble protein fractions were prepared from MJF373 cells transformed with either pTrcSdKefH6 or pTrcSdKefQCTDH6. Cells were culture overnight in LK medium (10 g/L tryptone, 5g/L yeast extract, 6.4 g/L KCl) with ampicillin (50 µg/mL) and diluted next morning to O.D.650 = 0.05 into a fresh LK medium as a preculture. Once cells had reached an O.D.650 of 0.4, they were diluted 10-fold into fresh LK medium and grown again until an O.D.650 of 0.4, when 0.3mM IPTG was added for induction of expression for 30 min, after which 100 ml cells were harvested by centrifugation, resuspended in PBS containing protease inhibitor cocktail tablet (Roche) and lysed by passage through a French Press at 18 000 psi. Bulk cell debris was removed by centrifugation for 10 minutes at 4 °C 4500 × g and

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membrane

(pellet)

and

soluble

fractions

(supernatant)

collected

after

ultracentrifugation at 90 000 x g, 4 C for 60 min. The pellet was suspended in PBS and Lowry26 estimation of protein concentration performed on the pellet and supernatant. Samples were separated on 4-12 % InvitrogenTM NuPAGE Bis-Tris gels (ThermoFisher Scientific) run in MES buffer and using SeeBlue Plus 2 Marker. Proteins were transferred onto nitrocellulose membranes and probed for expression using anti-His HRP conjugate antibody (Qiagen).

SuperSignal™ West Dura

Extended Duration Substrate (ThermoFisher Scientific) was used for ECL detection of bands, exposed to Amersham HyperfilmTM ECL film (GE Healthcare) developed on an M35 X-OMAT processor.

Expression and purification of the full-length protein SdKef The full-length membrane protein, was transformed into the E. coli strain BL21(DE3). Cells were grown the same as for SdKefQCTD. The cell pellet was resuspended in buffer 50 mM Tris-HCl buffer, pH 7.8, 300 mM KCl, 1 mM Benzamidine. After disruption of the cells with a French press at 18,000 psi, the suspension was centrifuged at 4 000 g for 20 min to remove cell debris. The supernatant was then centrifuged at 100 000 g for 1 h. The pellet which contained the cell membrane was solubilized in solubilization buffer 50 mM Tris-HCl buffer, pH 7.8, 300 mM KCl, 1 mM Benzamidine, 1.5% DDM, 10% glycerol, 25 mM imidazole by using a homogenizer and left for an hour gently shaking, at 4 °C. The solubilized sample was subsequently passed through a 25 mL column containing 0.5 mL of nickel-nitrilotriacetic acid (Ni2+-NTA) agarose, at 4 °C. The column was washed with 15 mL of wash buffer; 50 mM Tris-HCl buffer, pH 7.8, 300 mM KCl, 1 mM benzamidine, 0.05% DDM, 35 mM imidazole to remove non-specifically bound

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proteins and was left at 4 °C overnight. Next morning the elution followed with 10 mL of elution buffer; 50 mM Tris-HCl buffer, pH 7.8, 300 mM KCl, 0.05% DDM, 300 mM imidazole and 0.5 mL fractions were collected. The fractions were analyzed by SDS−PAGE and UV/vis absorption spectroscopy and the highest fractions were applied to a 120 mL Superose 6 column (General Electrics Healthcare) equilibrated with buffer containing 50 mM Tris-HCl buffer, pH 7.8, 300 mM KCl, 0.05% DDM. Protein was then eluted at 1 mL/min flow rate. Protein concentration was monitored by absorption at 280 nm. The column was calibrated with Biorad standards.

Structural biology SdKefQCTD was prone to aggregation over extended concentration required for structural studies, but using stirred ultrafiltration cell 8003 with a 30 kDa membrane cut off (www.millipore.com) and a nitrogen stream at 4 °C, prevented this, allowing a concentration of around 20 mg mL-1, in buffer containing 50 mM Tris-HCl, pH 7.8 and 300 mM KCl. Crystal trials were set up by hanging drop on freshly prepared protein samples that have not been previously frozen and involved mixing 1:1 and 2:1 volume of protein solution: precipitant equilibrated against a large volume of precipitant. Crystals grew to full size dimension of (0.2 mm × 0.05 mm × 0.05 mm) in two and a half months at 21 °C. The best crystals (judged by visual inspection) were obtained using 0.2 M sodium malonate pH 7.0 and 20% w/v PEG 3350, as precipitant. Prior to data collection, crystals were transferred into a solution containing 0.2 M sodium malonate pH 7.0 and 40% w/v PEG 3350. Data were collected at 100 K on a single crystal, which diffracted to a resolution of 2.92 Å on I24 at Diamond (Oxford, UK). Data were indexed, integrated and merged using MOSFLM / SCALA {Leslie, 1992, Joint CCP4 and ESF-EAMCB newsletter on

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protein crystallography, No 26, 1-10} as implemented in CCP4 {CCP4, 1994, Acta Crystallographica Section D, 50, 760-763}. The resolution limits were determined by the data statistics and the Wilson plot. The CCP4 program POINTLESS was used to assign space groups for Kef as P42212. The structure was solved using molecular replacement with the program PHASER using the E. coli KefC (PDB code 3EYW) as model containing residues 410 - 570 (omitting all water molecules and ligands with non-conserved residues set to alanine). AMP was modeled in both monomers and refinement proceeded by REFMAC5 and manual intervention COOT. Full crystallographic statistics are shown in (Table 1).

Table 1. Statistics of the SdKefQCTD X-ray crystal structure. Data collection Beamline Wavelength (Å) Resolution (Å) Cell constants (Å) α= β =γ=90 Unique reflections Mean I/σ Completeness (%) Multiplicity Rmerge Spacegroup Wilson B-factor (Å2)

KefQCTD from Shewanella denitrificans Diamond_I24 (21/10/2012) 0.9686 3.09 (47.6 - 3.09) a=71.6 b=71.6 c=140.4 6976 (399) 15.3 (1.9) 97.6 (85.8) 6.2 (3.3) 0.098 (0.651) P41212 77

Refinement R (%) Rfree (%) PDB accession code No of atoms Bond length rmsd Bond angle rmsd

20.53 (29.8) 26.04 (35.5) 5NC8 2448 0.01 1.558

Molprobity (S10) score centile

2.03 (99th centile)

Clashscore, all atoms, score centile

5.43 (100th centile)

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Ligand identification 100 µL of 200 µM purified SdKefQCTD in 50 mM Tris-HCl, pH 7.8, 300 mM KCl buffer and full length purified SdKef in 50 mM Tris-HCl, pH 7.8, 300 mM KCl, 0.05% DDM buffer, were denatured by being subjected to a constant temperature of 95 °C on a bench thermo block for 30 min. Total volume of sample containing the denatured protein was loaded on a vivaspin concentrator with a 3 kDa cut off and centrifuged at full speed on a bench top centrifuge until all liquid had gone through the membrane (30 mins). Filtrate was loaded on a Superdex Peptide 10/300 column (General Electrics Healthcare) with an optimum size separation from 100 to 7000 Da. Prior to sample loading, the column was equilibrated with buffer D. AMP, ATP and NADH of 100 µL volume and 2 mM concentration were individually loaded onto the same column on the same day, under identical conditions to calibrate its behavior. The UV active HPLC peak was subjected to MALDI mass spectrometry, as were the standards.

NMR NMR experiments were recorded at a 1H frequency of 600 or 700 MHz using a Prodigy BBO probe (600 MHz) or Bruker Avance III spectrometers equipped with a TCI inverse cryoprobe (700 MHz), respectively. The samples were prepared in 5 mm NMR tubes and experiments conducted at listed temperatures. CPMG experiments employed the PROJECT sequence (90°x − [τ − 180°y – τ - 90°y – τ - 180°y – τ]n – acq) as described by Aguilar et al. with a total filter time of 96 ms.27 In all edited 1H experiments water suppression was achieved by presaturation. The pulse tip angle calibration was done for all the samples using the Bruker pulsecal routine.

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Biochemistry

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The protein was prepared to a final concentration of 330 µM in deuterated sodium phosphate buffer as follows:

Exchange of protein solution with deuterated sodium phosphate buffer: The purified protein solution was exchanged with deuterated sodium phosphate buffer (50 mM NaH2PO4/Na2HPO4, 150 mM NaCl, pH 7.4, which were prepared in deuterium oxide) by using PD MiniTrap G-25 columns (GH Healthcare). The exchange was done by the spin protocol, according to the manufacturer’s instruction.

The reference spectra of each of the ligands also were run at a concentration of 330 µM in the deuterated sodium phosphate buffer: 50 mM NaH2PO4/Na2HPO4, 150 mM NaCl, pH 7.4, which were prepared in deuterium oxide.

Denaturation of protein for detection of AMP release: The protein was denatured on a heat block at 80 °C for 3 h. Subsequently, protein precipitate was centrifuged down and the resultant supernatant used directly for NMR studies. The ligands AMP, ADP and NADH were heat treated in the same manner as controls. The experiments were repeated without removing samples from NMR tubes to confirm AMP was not observed from contamination.

Native mass spectrometry Nanoelectrospray (nESI) MS experiments were carried out on a QToF mass spectrometer (Waters Corp., Wilmslow, UK) with conditions optimised for the transmission of intact noncovalent protein complexes.18 Sample was bufferexchanged into 200 mM ammonium acetate and sprayed at a concentration of 7.5 µM

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with respect to the dimer. Experiments were conducted in positive polarity with the following instrument settings: capillary voltage 1.4 kV, sample cone 40 V, extraction cone 25 V, backing pressure 3.5 mbar and collision cell pressure 1.75 MPa. The instrument was calibrated using CsI, and data analysis performed using MassLynx (Waters Corp., Wilmslow, UK) software. Protein masses were determined using the three most intense charge states.

Differential scanning fluorimetry Assays were performed using a Stratagene Mx3005P qPCR (Expt filter set, ex. 492 nm, em. 568 nm). The initial temperature was set to 25 °C (held for 5 minutes), increasing in increments of 1 °C for 55 cycles (held for 1 minute 30 seconds per cycle). Stock solutions of the ligands under examination were prepared to a concentration of 100 mM in buffer containing 50 mM Na-phosphate, 150 mM NaCl, pH 7.4 (with the exception of (−)-adenosine, which was prepared in DMSO). The 100 mM stock solutions were then diluted to a concentration of 10 mM in buffer containing 50 mM Na-phosphate, 150 mM NaCl, pH 7.4. A protein master mix was prepared containing SdKefQCTD (13.3 µM) and Sypro Orange (2.2×, Invitrogen) in buffer containing 50 mM Na-phosphate, 150 mM NaCl, pH 7.4. Ninety-six well plates (Axygen) were prepared using the protein master mix (22.5 µL per well; 12 µM final concentration of protein and 2× final concentration of dye) and the appropriate ligand (2.5 µL per well; 1 mM final concentration). The plate was centrifuged at 1000 rpm for 3 minutes before being run. Controls were performed with dye alone, ligand and dye, and the protein alone. The TM (melting temperature) was identified by fitting to the Boltzmann equation (Prism 5).19 The change in unfolding temperature (ΔTM) was calculated as the shift in TM relative to the TM of

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Biochemistry

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the protein + 2.5 µL buffer (DMSO in the case of (−)-adenosine), in the absence of any ligand. A Student’s t-test was performed to ensure that the changes were statistically significant.

Expression and purification of Kef mutant construct R416E SdKefQCTD To express the SdKefQCTD(R416E) the pTrcSdKefQCTDH6-R416E plasmid was first transformed into the E. coli strain, MJF373.12 The expression and purification conditions were the same as those employed in the alternative protocol for purifying the unmodified SdKefQCTD counterpart, except medium LK [10 g/L OxoidTM Tryptone (Thermo Fisher Scientific Inc.), 5 g/L OxoidTM Yeast Extract (Thermo Fisher Scientific Inc.), 6.4 g/L KCl (Thermo Fisher Scientific Inc.), 2 g/L glucose (Sigma-Aldrich Co.)] and cell mass of 6.26 g were used instead for protein expression and protein purification, respectively.

Quantification of bound AMP content in proteins by HPLC (R416E) Analytical HPLC was performed on a PerkinElmer Flexar system with a Binary LC Pump and UV/VIS LC Detector. A Dionex Acclaim® 120 column (C18, 5 µm, 120 Å, 4.6 × 150 mm) was used for analyzing the AMP content in unmodified SdKefQCTD as well as its mutant counterpart, SdKefQCTD(R416E). This HPLC analytic method had a constant flow rate of 1 mL/min and lasted for 20 min per run. It adopted a mobile phase with a mixture of Solvent A (99.9:0.1 : H2O:Formic acid) and Solvent B (99.9:0.1 : MeCN:Formic acid). This program employed 100% Solvent A at the first minute, then increased the concentration of Solvent B from 0 to 100% over 10 min using a linear gradient and this 100% Solvent B concentration stayed for further 3 min to the 14th min. After that, the method decreased the concentration of Solvent B to 0%

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at the 15th min, followed by 100% Solvent A running through the column for the last 5 min of the experiment. The whole HPLC program is summarized in Figure 1.

Figure 1. A summary of the HPLC program.

We adopted a similar experimental procedure developed by Chen and co-workers28 to analyze the bound AMP molecule in proteins. To release the bound AMP, proteins of defined concentrations (10 µL) were first heated at 95 °C in a dry heating block for 5 min, and then subjected to centrifugation at 13 000 rpm for 10 min (MIKRO 20 Centrifuge, Hettich) to pellet down denatured protein precipitates. The resultant pellet was resuspended in 10 µL SEC Buffer (50 mM NaH2PO4/Na2HPO4, 150 mM NaCl, pH 7.4) and the mixture was then centrifuged at 13 000 rpm for 10 min (MIKRO 20 Centrifuge, Hettich). The resulting supernatants from the above two centrifugation steps were combined (10 + 10 = 20 µL) and used as an injection sample for subsequent HPLC analysis. For the spiking HPLC experiments, supernatants from the first centrifugation step (10 µL) were combined with AMP solutions (10 µL) of defined concentrations.

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Computational methods Molecular dynamics (MD) simulations were performed using the SdKefQCTD model previously built by Healy and co-workers.1 A total of 4 systems were evaluated: a) SdKefQCTD with gluthathione (GSH) and 2 AMP molecules; b) SdKefQCTD with ESG and 2 AMP molecules; c) SdKefQCTD with GSH and d) SdKefQCTD with ESG. Molecular mechanics parameters for ESG, GSH and AMP were taken from the General AMBER Force Field (GAFF)2 with AM1-BCC atomic charges. Hydrogen atoms were removed from amino acid residues using the MolProbity Server2 and added using tLeap.3 Glutamate and aspartate residues were assigned as negatively charged and lysine and arginine as positively charged. Minimization and MD calculations were performed using the AMBER Force Field 12SB within AMBER version 12 with the GPU-accelerated version of PMEMD.4 Crystallographic waters were not removed, while the protein was further solvated by a box of TIP-3P water molecules.5 Simulations were carried out in octahedral boxes with an initial volume close to 160 nm3 containing 3,060 water molecules, adding counter-cations (Na+) to equilibrate the system. Energy minimization was performed in two steps. Firstly, we used steepest descent followed by conjugate gradients during which the initial position of the small molecule inhibitor and the protein structure obtained after homology modeling were restrained (PR). Secondly, the same minimization methodology was performed without PR. The minimized macromolecule:small molecule(s) complex was then subjected to 500 ps of equilibration and 40 ns of production MD simulation in the NPT ensemble using a Langevin thermostat to simulate a constant temperature at 310 K (τ T = 0.1 ps). Previous work performed by Zou6, Hong7, Shiao8 and Gewert9 and co-workers showed that nano scale MD could be enough to achieve reasonable protein models obtained by homology modeling.

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Isotropic position scaling was used to maintain the pressure of 1 atm (τ p = 2 ps).10 MD simulation was carried out using 1 fs of integration time and a nonbonding cutoff of 8 Å, with the Shake algorithm11 turned on to constrain bonds involving hydrogen. A total of 2 000 snapshots were obtained at intervals of 20 ps in producing plots of the geometric variation during the simulation.

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Biochemistry

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Results X-Ray crystal structure of the C-terminal domain of Kef from S. denitrificans (SdKefQCTD) A construct of S. denitrificans KTN carboxy-terminal domains (SdKefQCTD), was previously created to optimize stability and solubility.12 The protein was purified to homogeneity from E. coli BL21(DE3) and crystallized. So as not to bias the occupancy of the Rossmann fold, no nucleotide was added before, during or after purification or crystallization of the sample used for all crystallization trials. Therefore, the presence of AMP in the crystal has arisen from nucleotide binding inside the E. coli cell and subsequent co-purification. Optimization of initial sparse matrix crystallization conditions yielded a single crystal for which diffraction data were collected to 3.09 Å resolution (Table 1). The structure was solved by molecular replacement using the apo-KefC structure (PDB ID: 3EYW) by removing NAD+, as the searching model. The asymmetric unit contains two monomers that form the canonical KTN dimer. In line with the PISA17 prediction, gel filtration (Figure S1), size exclusion chromatography – multi-angle laser scattering (SEC-MALS, Figure S2) analysis, and analytical ultracentrifugation (AUC, Figure S3) experiments indicated that the two Kef monomers form a stable dimer in solution, at the concentrations used. The core fold of the SdKefQCTD domain is essentially identical to that previously described for the E. coli protein.10,16 Each monomer has 6 β-strands (b1-6) arranged in a parallel sheet, which is sandwiched between three α-helices on one face and 1 α-helix on the other. Two C-terminal helices (a5, a6), resolved in the structure, form a helix-turn-helix type arrangement and reach across to the other monomer. The penultimate helix (a5) pairs with a single helix (a1) from the other

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monomer and stacks against the β-sheet from the other subunit. The long C-terminal helix (a6) makes contacts with both monomers (Figure 2A). A

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B Figure 1 A) KefQCTD from Shewanella denitriﬁcans forms a dimer with each one of nucleo

Adenosine Monophosphate Binding Stabilizes the KTN Domain of

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